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Structure of Spindle Associated Protein Spindlin1 in Complex with Methylated Histone

Author: WangYan
Tutor: GuoShiHua；XuRuiMing
School: Inner Mongolia Agricultural University
Course: Crop Genetics and Breeding
Keywords: spindlin1 protein Subcloned Protein expression and purification Protein crystals Crystal structure
CLC: Q51
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract

In this paper, structural biology means the spindle related protein spindlin1 of its methylated histone peptide substrate structure of the complex formed. Using a large number of prokaryotic cell expression of recombinant proteins in vitro constructed spin gene prokaryotic cells the expression vector pET28a/SUMO and pGEX-2T, according to the expression results, it was confirmed the soluble recombinant spindlin1 can be obtained a large number of tag and GST tagged with a his-SUMO. Cultured in LB medium, E. coli strain BL21 (DE3) is carried out in large-scale expression of the target protein. Electrophoresis test results show that the ideal recombinant protein can be routinely cultured and induced conditions (37 ° C culture; 16 ℃, 0.3 mmol / L IPTG was inducible expression). Separation and purification of the target protein using a column chromatographic methods, and established stable purification strategy can be repeated, i.e. the affinity chromatography → anion exchange chromatography → gel filtration chromatography to obtain a crystalline pure, high concentrations (about 24mg/mL or 0.9 mmol / L) The purpose of the protein solution, and used for crystallization experiments. With the purpose of the purified protein product The selection and optimization of crystallization conditions, and ultimately, to establish a stable, repeatable and capable of forming a high-quality crystals of the crystallization conditions, i.e.: 0.1 mol / L, CAPS, pH = 11; 2.0 mol / L (NH < sub> 4 ) ₂ SO ₄; 0.2mol / L Li ₂ SO ₄; 0.1 mol / L Glycine, obtained protein crystals suitable for diffraction experiments. Synchrotron radiation light source in Shanghai, using X-ray diffraction analysis of protein complexes crystals collected to a resolution of 2.4? Diffraction data, which preliminary analysis and correction H3K4me3 peptide complexes spindlin1 and its binding substrates The three-dimensional structure. Detailed information on both interaction is obtained by the structure model, i.e.: the second Tudor spindlin1 domains involved in the recognition and binding of trimethylated a lysine residue, wherein the amino acid residue positions spindlin1 critical point is (starting from the N-terminal) Phe116, Trp126, Tyr145, Tyr152 and four with the residue of the aromatic ring; spindlin1 the residue Tyr154, and tri-methylation of lysine residues stabilized through hydrogen bonding between interaction; the Tudor domain of spindlin1 second residues Asp159 and groups of residues in the protein polypeptide Arg2 salt bond between the stable interactions; the histone H3K4me3 polypeptide residue Arg8 or methylation occurs residues Arg8 may of extending into spindlin1 first the Tudor structure domains to enhance the stability of the overall recognition and binding force.

Structure of Spindle Associated Protein Spindlin1 in Complex with Methylated Histone

Abstract

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